Book/Dissertation / PhD Thesis

FZJ-2018-02970

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Expression, purification and biophysical characterization of human Presenilin 2

Yang, G. (Corresponding author)FZJ*

2013
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag Jülich
ISBN: 978-3-89336-928-7

Please use a persistent id in citations: http://hdl.handle.net/2128/18547

Abstract: Presenilin proteins are part of the γ-secretase complex which consists of four different membrane proteins. This complex plays an important role in the development of Alzheimer's disease. The cleavage of APP-C99 by $\gamma$-secretase generates the amyloid beta peptide which is the major component of amyloid plaques. Presenilins are considered to be the catalytic subunit of $\gamma$-secretase. They are activated by auto-cleavage into its N-terminal and C-terminal domain to act as a protease within the membrane. Structural information of presenilins and the $\gamma$-secretase are of great importance to understand the activity of the complex and its mechanism of action. In the presented work, human PS2 full length (PS2-FL) and its N-terminal domain (PS2-NTF) as well as its C-terminal domain (PS2-CTF) were heterologously expressed in $\textit{E.coli}$ via $\textit{in vivo}$ or by $\textit{in vitro}$ expression in order to characterize the proteins and to obtain material of sufficient quantity and quality for structural investigation via X-ray crystallography or NMR spectroscopy. By optimization of the expression conditions and using codon-usage optimized DNA, PS2-FL and PS2-NTF can be expressed with a level of about 1 to 2 mg per liter culture via in vivo expression. Purification of PS2-FL was performed under native and denaturing condition by a combination of affinity chromatography and SEC. SEC analysis revealed that PS2-FL exists in equilibrium of hexamer and trimer. The observed tendency of the protein to aggregate, especially material obtained by refolding, appears to be caused at least partially by instability of the tertiary structure. A structural instability of a tertiary structure of a protein whose domains will be part of the quaternary structure of a protein complex after cleavage may be simply caused by flexibility of the domain assembly in the absence of other proteins of the complex. Indeed, the functional domains of PS2 show different oligomerisation than the full-length protein. CD and fluorescence spectroscopy measurements of the refolded His-PS2-FL reconstituted into MO cubic phase revealed that the protein still exhibits the characteristics of a predominantly α–helical protein and the localisation of the tryptophan side chains in a relatively non-polar environment. Therefore first [...]

Keyword(s): Preseniline ; Genexpression

Note: Universität Düsseldorf, Diss., 2012

---

Contributing Institute(s):

1. Strukturbiochemie (ICS-6)

Research Program(s):

1. 899 - ohne Topic (POF3-899) (POF3-899)

---

Database coverage:

---